Technical Field Text
Diagnosis of disease is made by matching current observations to established correlations of past observations to the known clinical outcomes. For a disease such as cancer, clinical diagnosis is most often made by taking a sampling of cells or tissues from an organ or region of the body and examining the specimen using an optical microscope. Many observations are made, such as the tissue structure, cell morphology, and subcellular morphology and chromatin distribution in the nucleus. To obtain samples of the cells and/or tissue, a biopsy is often taken.
There are many different ways to take a biopsy specimen. Open surgical techniques allow direct access to tissue so specimen size does not have to be limited. Minimally invasive techniques impart less trauma to the healthy tissues of the body, but specimen size per biopsy is usually limited. Minimally invasive biopsy tools for tissue sampling range from surgical cuttings and punches, forceps, and coring needles. Less invasive and typically smaller biopsy tools are used for cell sampling, which include fine-needle aspiration, brushing, and tissue washes.
Tissue biopsy is generally preferred over cell biopsy because tissue architecture is preserved in the tissue biopsies. Although disease diagnosis such as cancer can be made with individual cells, the tissue architecture provides additional information about the location and extent of the disease. The invasiveness of cancer can be determined from tissue biopsy rather than cell biopsy, which affects the treatment of the patient.
Needle biopsy can take either cells or tissue. The largest needles have sharp tips that pierce the tissue and then side chambers that cut the tissue pressing against the needle shank. These side-cutting needles are used to take 1-4 mm diameter cores of tissue, which are often a couple centimeters long. Needles that have cutting tips can also take a core of tissue. These forward-cutting needles can be smaller in diameter, but below 0.5 mm inner diameter, the needle is not able to reliably remove the core specimen from the body. Thus, core needles are typically larger than 0.5 mm inner diameter.
Once a core of tissue is taken from the body, the specimen is handled in similar fashion to all tissues removed for disease diagnosis. The tissue is chemically fixed and stained with absorptive dyes. Typically hematoxylin stain is used to make the nuclear structure blue in color, while eosin is used to stain the cytoplasmic structure pink. To observe the stained tissue structure at sufficient spatial resolution, an optical microscope is used in transmission. However, tissue attenuates transmittance of white light, primarily due to scattering from refractive index differences of the structures in the tissue. Since the optical microscope is limited to tissue thicknesses of less than 0.1 mm, thin sections of the biopsy specimen are cut in an orderly fashion to represent the three-dimensional (3D) tissue architecture from the two-dimensional (2D) images.
Thinner needles are less invasive, but are not used to reliably acquire a tissue specimen. The thin needle is used to acquire cells dislodged and disassociated from the tissue. The biopsy of aspirated cells from these thin needles is called a fine needle aspirate (FNA). The FNA specimen is most often analyzed cytologically, as individual cells spread on an optical microscope glass slide and observed at higher optical magnification than tissue specimens. Often the exact same types of absorptive stains are used to color the cell components. However, a different chemical fixative is often used in cytology, which better preserves the chromatin structure.
In summary, the smaller and less invasive needles acquire FNA specimens, which consist of isolated cells in a slurry, while larger needles are used to acquire core tissue specimens. Between these needle sizes, core tissue specimens are acquired occasionally. For example, needles of 22-gauge or 0.4 mm inner diameter can acquire core specimens in approximately 3 out of four cases, as reported by C. Jenssen and C. F. Dietrich (2009) “Endoscopic ultrasound-guided fine-needle aspiration biopsy and trucut biopsy in gastroenterology—an overview,” Best Practice & Research Clinical Gastroenterology, 23: 743-759. The sizes of biopsy needles are listed by gauge, which is converted to inner diameter in millimeters below.
Inner diameters of needles and their medical useInner diameterGauge (G)(ID) mmMedical use112.4core141.6core161.2core190.7core210.5core/FNA220.4FNA250.2FNA
In the above example of endoscopic ultrasound-guided FNA biopsy, the advantage of the thinner needle is two-fold. (1) The smaller needle can be fit more easily through the working channel of a flexible endoscope that reaches the pancreas by passing through the mouth, throat, and stomach. Thus, the endoscope must have tight curves that restrict larger-sized needle devices from reaching all regions of the pancreas. For example, only the thinner needle devices, such as FNA, can be used to biopsy from all regions of the pancreas using conventional flexible endoscopes. The FNA needles are typically 5× thinner than the standard core needle. (2) The smaller needle is less invasive, which is much more important for delicate organs such as the pancreas, as pancreatitis is life threatening. Thinner needles are very useful in other organs like the brain.
However, the advantage of the core needle biopsy is the more valuable tissue specimen. Tissue specimens are preferred for being able to better determine the extent and invasiveness of disease, such as cancer.
Thin Needle Core Biopsy (TNCB)
Even though engineering improvements in thin-needle coring devices can produce finer core specimens, such as 0.25 mm in diameter, the pathologist has no established procedure to make a diagnosis from a less invasive tissue biopsy. A thin-needle core biopsy (TNCB) of 0.25 mm diameter by one to two centimeters in length is too small and fragile of a tissue specimen to handle. The cell-to-cell bonds holding together diseased tissue such as cancer are often much weaker than normal tissue. Manual techniques used for the conventional core needle biopsy specimens of roughly 5× to 10× diameter cannot be used without damaging the tissue structure. Any TNCB specimen that is sub-millimeter in diameter is expected to be too small for the traditional method of cutting thin slices in an orderly fashion to determine extent and invasiveness of disease.
Although TNCB specimens that are sub-millimeter in diameter are considered too small mechanically, these same specimens are considered too thick to make a straightforward optical diagnosis. Because white-light light transmission through tissue is usually limited by optical scattering to less than 0.1 mm, TNCB specimens greater than this diameter cannot be imaged directly on a conventional microscope used by pathologists. Thus, TNCB specimens in the range of 0.1 to 1 mm in diameter are problematic for making disease diagnosis, considered too small for conventional sample handling and preparation, while also considered too large for routine optical imaging for making a diagnosis. Thus new techniques in both specimen preparation and imaging are necessary to use the lease invasive tissue biopsy sample for disease diagnosis.
Advanced methods for small-sized sample preparation are still inadequate. More automated devices for acquiring core biopsy specimens from a needle has been proposed which can reduce manual handling requirements. For example, a core needle biopsy device has been proposed that has a specimen collection and retention chamber where the specimen can have applied fluid and vacuum for processing the specimen by Quick et al., (2009) in U.S. Pat. No. 7,572,236, entitled, “BIOPSY DEVICE WITH FLUID DELIVERY TO TISSUE SPECIMENS.” However, human handling is still required for histological imaging since there is no mechanism for further specimen handling before diagnostic imaging.
Automated cell handling and in vitro diagnostics have been proposed for isolated cells and sub-cellular constituents, but there has been little advancement using this technology for larger multicellular specimens like TNCB. The first use of a microfluidic system for human biopsy tissue samples for histopathological diagnosis used a large tissue slice where smaller microfluidic chambers were placed on top of the tissue, see Kim et al., (May 2010) in PLoS ONE online journal (volume 5, issue 5, e10441), entitled, “Breast cancer diagnosis using a microfluidic multiplexed immunohistochemistry platform.” The resulting diagnosis by optical imaging was made from these individual small chambers that provided diagnostic sampling from the single large tissue specimen. Biopsy specimens, such as TNCB, are not inserted into a microfluidics device for automated specimen preparation and diagnosis for the entire biopsy specimen.
Microfluidics was first developed in the 1980s as a means for precisely manipulating fluids. The field has been expanded significantly to biological applications through major university research, such as Yager Lab at the University of Washington (http://faculty.washington.edu/yagerp/, accessed 19 Apr. 2011) and even spread to industrial applications at companies such as Micronics (http://www.micronics.net/, accessed 19 Apr. 2011).
Techniques for optical imaging biopsy specimens are inadequate for TNCB specimens. Typically biopsies from thin needles of <0.1 mm are generated into a slurry of cells, such as aspirates (e.g. FNA). These specimens have lost most or all of their cell-to-cell bonds and tissue architecture is lost. The methods employed to image these isolated cells and tissue fragments consist of near monolayers of cells to smears of cells on a microscope slide for standard image analysis by a cytologist. These cell samples can also be analyzed using flow cytometers, imaging flow cytometers, and the optical projection tomography microscope, see Fauver et al., (2005) “Three-dimensional imaging of a single isolated cell nucleus using optical projection tomography,” Optics Express 13(11): 4210-4223.
Tissue biopsies that are larger have a wider array of techniques used for optical imaging, although over 90% of all cancer diagnosis is performed using thin sections of tissue that is stained for conventional bright field optical imaging using white-light in transmission. The more advanced techniques for optical imaging are moving optical diagnosis from reliance on 2D images to more three-dimensional images. These techniques range from laser scanning confocal, multiphoton excitation, to new super-resolution optical imaging. However all these techniques rely on fluorescence marking of tissue structures. Thus there is a gap between these research microscopes and clinical diagnosis which relies not on fluorescence, but on absorptive stains that are imaged with white light that is transmitted through the tissue.
Alternative imaging techniques such as optical coherent tomography (OCT), holographic imaging, and enhanced backscattering techniques rely not on the absorption of light from stained tissue structures, but on the scattering of light from unlabeled structures. Because there is no correlation of these clinical outcomes to new microscopic images of tissue and cells, there is no basis for making routine clinical diagnosis of disease. This same problem holds for new optical techniques that use chemical signatures of cell and extracellular structures to form images, such as coherent Raman scattering.
A technique that has produced 3D images of tissue that has direct clinical relevance is from Sharpe et al. (2002) “Optical projection tomography as a tool for 3D microscopy and gene expression studies,” Science 296, 541-545. Optical projection tomography (OPT) uses a narrow beam of light that has a large depth of focus, which is on the length scale of the tissue specimen thickness. This beam of light is scanned through the tissue as the tissue orientation is changed (i.e., specimen is rotated). The resulting series of optical projection images are created in transmission so standard absorptive stains of hematoxylin and eosin can be used. The series of 2D images can be used to create a 2D image using a 3D reconstruction mathematical technique that is similar to x-ray computed tomography.
Advantages of 3D Imaging for Disease Diagnosis
Imaging tissue in 3D is advantageous over one or more images in 2D for several reasons. The original object of cells and tissue are three-dimensional and the human brain is trained to interpret 3D objects in their natural state. A 3D image and 3D visualization can provide clear localization of the disease and surrounding tissue without ambiguity from overlapping structures. The extent of disease can be clearly assessed and regions of interest measured. Invasiveness of the suspected disease can be tracked through the surrounding tissue. Surgery and treatment regimens can be better planned for the patient. The advent of 3D computed tomography (CT) using x-rays has replaced standard 2D x-rays for many medical applications due to these reasons.
For the case of TNCB specimens, there are advantages to imaging in 3D rather than 2D. The advantage of imaging tissue rather than dissociated cells is that more than simply the presence of disease, such as cancer, can be made from the tissue specimen. The location of the cancer cells or tumor, extent of disease, and most importantly the invasiveness of cancer can be ascertained. For example, determining if cancer has migrated from the epithelium through the basement membrane to the capillary beds is extremely important information for treating the patient and managing the disease.
The advantage of 3D imaging the entire TNCB specimen is that there are no sampling errors. Unlike conventional core needle biopsies of 1 to 3 mm in diameter, the entire specimen is too large to optically image the entire specimen, so thin slices must be taken at different locations. There is risk of missing small tumors and especially early cancers. Since the TNCB specimens of 0.1 to 1 mm are smaller they can be optically imaged in their entirety in 3D. Again, 2D imaging would take only a cross-sectional sampling of the 3D specimen, although this is currently the standard clinical practice.
3D imaging of biological specimens has been accomplished in the transmission mode, which allows the use of clinically important absorptive stains. Two similar techniques of Ohyama et al., (1997) in U.S. Pat. No. 5,680,484 entitled, “OPTICAL IMAGE RECONSTRUCTING APPARATUS CAPABLE OF RECONSTRUCTING OPTICAL THREE-DIMENSIONAL IMAGE HAVING EXCELLENT RESOLUTION AND S/N RATIO,” and Nelson (2007) in U.S. Pat. No. 7,197,355 entitled, “VARIABLE-MOTION OPTICAL TOMOGRAPHY OF SMALL OBJECTS,” never disclose a means for forming a continuous image of a tissue specimen along the longitudinal axis of the rotating tube. Whereas, Sharpe & Perry (2007) in U.S. Pat. No. 7,218,393 entitled, “ROTARY STAGE FOR IMAGING A SPECIMEN,” imaged a single specimen using a focused beam of light through the rotating specimen within a stationary chamber with sidewalls that are orthogonal to the optical axis. The main shortcoming of this OPT technique is the low spatial resolution which is defined by the size of this focused beam of light, see Miao et al., (2010) “Resolution improvement in optical projection tomography by the focal scanning method” Optics Letters 35(20): 3363-3365. In addition, there is a problem with this technique for rotating long, thin, and fragile tissue specimens to form 3D images since no transparent containment tube is used.